Method for affecting phenotypic activity of endophytic fungi

ABSTRACT

Described herein are methods for the alteration and/or transfer of fungal functional traits, e.g., phenotypic activity, via the controlled transfer of endohyphal symbionts, e.g., bacteria, among fungal species. Also described are methods for the identification of endohyphal bacterial symbionts as determinants of cellulase and ligninase activity in fungi, and the use of endohyphal bacterial symbionts to alter the activity, including cellulase and ligninase activities, of the fungi. In particular, the fungi described herein are endophytic fungi, that is, fungi which colonize living, and subsequently senescent, plant tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Nonprovisional application Ser.No. 14/745,365 filed on Jun. 19, 2015, and claims priority to U.S.Provisional Application No. 62/014,615 filed on Jun. 19, 2014, theentire contents of which are incorporated herein for all legal purposes.

GOVERNMENT FUNDING

This invention was made with government support under Grant Nos.DEB1045766, DEB0702825, and IOS1354219 awarded by the National ScienceFoundation (NSF). The government has certain rights in the invention.

FIELD OF THE INVENTION

Plants and their associated microorganisms, for use in medical,industrial, agricultural, horticultural, forestry and otherapplications.

BRIEF SUMMARY OF THE INVENTION

Described herein are methods for the alteration and/or transfer offungal functional traits, e.g., phenotypic activity, via the controlledtransfer of endohyphal symbionts, e.g., bacteria, among fungal species.Also described are methods for the identification of endohyphalbacterial symbionts as determinants of cellulase and ligninase activityin fungi, and the use of endohyphal bacterial symbionts to alter theactivity, including cellulase and ligninase activities, of the fungi. Inparticular, the fungi described herein are endophytic fungi, that is,fungi which colonize living, as well as senescent, plant tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is graph showing the results of in vitro mass-lossexperimentation on fresh and senescent tissue of Juniperus deppeana andCupressus arizonica as a function of treatment with seven fungi with (+)and without (−) EHB.

FIG. 2 is a graph showing the mean colony diameter of Pestalotiopsis sp.9143, Microdiplodia sp. 9145, and axenic bacteria during cellulaseassays as a function of EHB (Luteibacter sp. 9143 or Luteibacter sp.9145) and type of association.

FIG. 3 is a graph showing the average colony diameter of Pestalotiopsissp. 9143 or Microdiplodia sp. 9145 on indulin medium as a function ofbacterial strain (Luteibacter sp. 9143 or Luteibacter sp. 9145) and typeof association.

FIG. 4 is a graph showing the results of in vitro mass-lossexperimentation on P. orientalis foliage, including (A) fresh, greenleaf material and (B) senescent leaf material treated with each of sevenfoliar fungi with (+) and without (−) EHB.

FIG. 5 is a graph showing in vitro mass loss from foliage of P.orientalis as a function of foliage state (fresh, senescent) and EHBstatus for each fungus.

FIG. 6 is a graph showing the relationship of mass loss vs. the visualscore of fungal growth, with the 95% confidence interval for the linearfit shaded along the best-fit line.

DETAILED DESCRIPTION OF THE INVENTION

Plant-associated fungi provide important ecosystem functions aspathogens, mycorrhizae, endophytes and saprotrophs. Endophytic fungicolonize living plant tissues in all biomes, often providing plants withprotection from pathogens, herbivores and other environmental stressors.As saprotrophs, fungi are the primary decomposers of senescent plantmaterials, cycling nutrients by breaking down cellulose and lignin, themaj or components of plant cell walls. Many fungi play more than one ofthese roles throughout their life cycles.

Endophytic fungi have been found to harbor (i.e., host) endohyphalbacteria (referred to herein as “EHB”). EHB live within apparentlyhealthy, viable fungal cells. EHB of foliar fungal endophytes were firstdocumented in endophytes of cupressaceous plants (Hoffman and Arnold2010—Hoffman, M. and A. E. Arnold. 2010. Diverse bacteria inhabit livinghyphae of phylogenetically diverse fungal endophytes. Applied andEnvironmental Microbiology 76: 4063-4075.) EHB in endophytes aredistinct from bacterial endosymbionts of other plant-associated fungi,are phylogenetically diverse and can live outside their fungal hosts.

EHB can have strong effects on fungal phenotypes, influencing theinteractions between fungal endophytes and the host plants of the fungalendophytes and the enzymatic and other activities of fungi in culture.

Described herein are methods for influencing plant-fungi interactionsutilizing EHBs, based on the inventor's determination that the presenceand identity of EHB significantly influences the success of plantinfection by endophytic fungi, and that these effects display somehost-specificity.

Also described herein are methods for influencing the production ofcellulase and ligninase enzymes by fungi via the removal, introduction,or exchange among fungi of EHBs.

Further described herein are methods for the alteration and/or transferof fungal functional traits, e.g. phenotypic activity, via thecontrolled transfer of endohyphal symbionts among foliar endophyticfungal species.

The methods described herein provide a mechanism for EHB obtained from afirst fungus that is infected with that EHB, to be transferred to asecond fungus of a different species than the first fungus.

Further, the methods described herein provide a mechanism to transferEHB from a first fungus to a second fungus, wherein the second fungus isof a different class than the first fungi. For example, the first fungicould be of the class Dothideomycetes (e.g., Microdiplodia) and thesecond of the class Sordariomycetes (e.g., Pestalotiopsis).

The methods described herein can be applied to numerous other foliarendophytic fungi and EHB combinations. In particular, the methodsdescribed herein appear to be particularly suitable for transfer amongstspecies and classes within the phylum Ascomycota. The phylum Ascomycotais the most species-rich phylum of fungi. It includes diverse plantpathogens, animal pathogens, and species that produce medicinal productsand pharmaceuticals, industrial products, and agents of biologicalcontrol. Examples of re-synthesized or re-associated fungal-EHB “pairs”are shown in Table 1. For example, the fungus Microdiplodia 9145 isre-synthesized with EHB Luteibacter sp. 1.

Also described herein are methods for transferring endohyphal bacteriabetween two endophytic fungi of different species and/or differentclasses, comprising cross-innoculating each fungus with the bacterialinoculum of the other fungus. More specifically, a first bacterialinoculum of the EHB bacteria from a first fungus is prepared, and asecond bacterial inoculum of the EHB bacteria from a second fungus isprepared, and then the first fungus is inoculated with the second EHBinoculum, and the second fungus is inoculated with the first EHBinoculum. The endophytic fungi may be of the same species, or may be ofdifferent species, or may be of different classes. The endohyphalbacteria from each fungus may be of the same species, or may be ofdifferent species, or may be of different classes or phyla/divisions.

Methods for examining phenotypic changes in endophytic fungi associatedwith bacterial symbiosis are described, wherein the fungi are “cured” oftheir endohyphal bacteria, and thereafter the activity of the fungi isexamined and compared to the activity prior to “curing”.

Another method involves “curing” the fungi of their endohyphal bacteria,and then inoculating the fungi with the same or different species ofbacteria (to re-synthesize the symbiosis), and thereafter examining theactivity of the fungi and comparing their activity from their nativestate, to their cured state, to their re-synthesized symbiotic state.

Also described are methods for the identification of endohyphalbacterial symbionts as determinants of cellulase and ligninase activityin fungi, and the use of endohyphal bacterial symbionts to alter theactivity, including cellulase and ligninase activities, of the fungi.

Still yet another method described herein is a method for altering thephenotype(s) of endophytic fungi by “curing” and/or transferringendohyphal bacteria from a first fungus to a second fungus.

For example, the phenotype that is altered may be the enzymatic activityof the endophytic fungi. The enzymatic activity may be increased ordecreased. Examples of enzymatic activity are ligninase and cellulaseactivity. The methods of the invention could be used in a variety ofapplications, such as those relevant to biofuels development from plantmaterial, environmental remediation, etc.)

EHB presence can affect cellulase and ligninase production of fungalendophyte cultures. The effect appears to result not from enzymeproduction by the EHB but rather from the symbiosis of the EHB and thefungi, as illustrated by different results discussed herein when thesame bacterium was present in different fungal taxa. Also, fungalcultures infected with EHB can increase degradation of plant material ofnative hosts.

Alternatively, the phenotype altered may be the growth rate of theendophytic fungi. Fungal growth rate can be altered by EHB presence.Growth rate of the fungi infected with EHB may be further enhanced orslowed when exposed to different nutrient and temperature conditions.EHB presence may restore fungal growth in an otherwise inviableenvironment.

Further, EHB can influence plant infection of fungal endophytes.Depending on the bacterium, different EHB can aid or inhibit fungalinfection in closely related endophytes.

The following is an embodiment of a method according to the invention,wherein EHB is obtained from a first fungi, a second fungi is “cured” ofits own naturally occurring EHB, and the EHB obtained from the firstfungi is re-synthesized (i.e., re-associated) with the second fungi.

1. A fungal endophyte culture naturally infected with EHB is produced asfollows: a surface-sterilized leaf tissue is cut into 2-mm² pieces andplated on nutrient media (2% Malt Extract Agar);

2. An EHB is obtained from the naturally-EHB-infected fungal endophyteculture as follows: a portion of the first fungal endophyte cultureproduced in Step 1 is incubated at 36° C. to induce bacterial emergence,and then the bacteria are streaked onto Luria Broth Agar (LBA); bacteriaare grown in 5 mL LB at 36° C. and 200 rpm for 2 days, rinsed twice with10 mM MgCl₂, resuspended in 2.4% PDB, quantified with a spectrometer andnormalized to a final volume of 3 ml;

3. A fungal endophyte culture cured of EHB is produced as follows: aplug of mycelia from a second (“second” refers to the fungal-endophytebeing of a different species and/or class from the first fungalendophyte used in Step 2 above) fungal endophyte culture naturallyinfected with EHB is transferred to fungal media amended with antibiotic[2% MEA amended with Ampicillin (100 μg/ml), Kanamycin (50 μg/ml),Tetracycline (10 μg/ml) and Ciprofloxacin (40 μg/ml)];

4. The fungal endophyte culture cured of EHB is then blended in 100 mL2.4% Potato Dextrose Broth, and grown at 27° C. and 100 rpm for 10 days;

5. The fungal endophyte culture that has been cured of EHB (produced inStep 3) is then re-synthesized with EHB, by co-culturing it with the EHBproduced in Step 2.

The following is an alternative embodiment of a method according to theinvention. In this example, fungi Pestalotiopsis 9143 is re-associatedor re-synthesized with Luteibacter 9143, and the step of curing thesecond fungi of its natural EHB infection is not described.

Culture Fungal Symbiont According to Steps 1-7, as Follows:

1. Blend a plug of mycelium (1¼ cm diameter) from inside the edge of anactively growing fungi colony (grown on 2.4% Potato Dextrose Agar) in100 mL 2.4% Potato Dextrose Broth for 5 sec. on low 3×, transfer tosterile flask;

2. Incubate at 27° C. and 100 rpm for 10 days;

3. Collect mycelium via vacuum filtration onto a #2 Whatman filterpaper;

4. Rinse mycelium with sterile water, scrape mycelia off filter paperwith sterile forceps and resuspend in 2.4% PDA;

5. Blend as in step 1;

6. Quantify with a spectrophotometer (OD 600); and

7. Transfer desired amount of inoculum to sterile flasks, inoculateimmediately with bacteria.

Culture Bacterial Symbiont, According to Steps 8-12, as Follows:

8. Inoculate 5 mL Luria Broth with a bacterial colony, vortex;

9. Incubate at 36° C. and 200 rpm for 3 days;

10. Centrifuge cultures at 300 rcf for 3 minutes, discard supernatant,and rinse with 4 ml of sterile 10 mM MgCl₂. Repeat wash;

11. Re-suspended bacteria in 4 ml 2.4% PDB; and

12. Quantify with a spectrophotometer (OD 600) and normalize to a finalvolume of 3 ml.

Co-Culture Symbionts According to Steps 13-17, as Follows:

13. Add 3 ml of the resuspended bacterial culture (EHB) to flasks withmycelium (fungi). The ratio of mycelium:bacteria should be 5:1 beforeinoculation;

14. Incubate at 27° C. and 100 rpm for 7 days;

15. Transfer 200 μL of co-culture to the center of water agar plates;

16. Incubate at 27° C. for 2 weeks; and

17. Screen the growing edge of fungal colony for EHB as described inHoffman and Arnold, 2010 (Hoffman, M. and A. E. Arnold. 2010. Diversebacteria inhabit living hyphae of phylogenetically diverse fungalendophytes. Applied and Environmental Microbiology 76: 4063-4075

Bacterial Screening to Determine Whether Fungal Isolates (i.e., Fungi“Cured of EHB) were in Fact Free of EHB.

Genomic DNA of fungal isolates were screened for bacterial infection by16S PCR using RedTaq (Sigma) and rDNA primers 27F/1492. Positive PCRproducts were cleaned using ExoSAP-IT, Sanger-sequenced bidirectionallyat the University of Arizona Genetics Core. Sequences were assembledautomatically and basecalls made byphred and phrap with orchestration byMesquite, followed by manual editing in Sequencher and BLAST comparisonswith GenBank (U'Ren et al. 2010—U'Ren J M, Lutzoni F, Miadlikowska J,Arnold A E (2010) Community analysis reveals close affinities betweenendophytic and endolichenic fungi in mosses and lichens. Microb Ecol60:340-353). Cultures with positive 16S products were examined forextrahyphal bacteria and viability of EHB with Invitrogen's LIVE/DEADBacLight Bacterial Viability Kit. Negative PCR products were cloned(Agilent, StrataClone) to provide further evidence for absence ofbacteria.

Ligninase and Cellulase Activity Assays.

Various cellulase and ligninase assays were conducted to show the effecton the production of cellulose and ligninase by the foliar endophyticfungi and depending upon the EHB. EHB Luteibacter inhibited theproduction of ligninase in the endophyte Microdiplodia 9145 and aidedthe production of cellulase. Endophyte Pestalotiopsis 9143 also harborsa bacterium from the same genus but it did not degrade celluloseregardless of infection status, and the production of ligninase was notaltered with infection. Cellulase production of Alternaria 9055 wasdecreased by infection of Sphingomonas; however, production was enhancedfor Cladosporium 9128 when infected with Curtobacterium. There was noeffect of EHB on enzyme production in four of the tested endophytes.

For each fungal endophyte/EHB infection status, six petri dishescontaining equal volumes of 2% Malt Extract Agar with 0.5%carboxymethylcellulose for cellulase assays (C) or water-agar with 0.05%indulin for ligninase assays (L) were each inoculated with a 6 mm plugof actively growing mycelium. Inoculated plates were incubated at 22° C.until the diameter of the colony reached 3-4 cm then flooded with 0.2%w/v Congo red (C) or with a 1.0% w/v FeCl₃ and K₃ [Fe(CN)]₆ solution(L), incubated at room temperature for 40 min. and washed several timeswith a 1M solution of NaCl (C) or tap water (L) (Gazis et al. 2012).Fungal colony diameter and zone of clearing were then measured over twoaxes then averaged.

In Vitro Mass-Loss Experiment.

Dry plant material of Platycladus orientalis treated with EHB infectedendophytes lost more mass and displayed more fungal growth than materialtreated with EHB-free endophytes Platycladus orientalis is the originalhost of the experimental endophytes. There was no statisticallysignificant difference between fresh and dry material inoculated withEHB+/− fungal endophytes in that plant species, or in J. deppeana or C.arizonica.

Fresh (green) and dry (brown) leaf material was collected for threespecies of Cupressaceae: Platycladus orientalis, Cupressus arizonica andJuniperus deppeana, from branches ˜1.5 m above ground at the Universityof Arizona Campus Arboretum. Tissue was placed in 100 mm Petri dishes(three replicate plates for each plant species/tissue type/fungalendophyte/infection status) and surface sterilized by rinsing with tapwater then flooding three times with 95% EtOH (10 sec), 10% Bleach (2min), and 70% EtOH for (2 min) (Arnold 2002). Each plate received 3 mLof sterile water and was inoculated with 75 uL of fungal inoculumprepared by grinding a 6-mm plug of actively growing mycelium (nomycelium added to the negative control) in sterile water. Plates wereheavily Parafilmed, weighed immediately, then weekly for 3 weeks atwhich time fungal growth was scored from 0-4 (“zero” means no visiblegrowth and “4” means 76-100% fungal growth.) Total mass loss was scaledby original weight and then by the average of the controls.

Plant Inoculations.

Three cupressaceous plants (Platycaldus orientalis, Cupressus arizonicaand Juniperus deppeana) were inoculated with fungal suspensions preparedwith one plug (6 mm in diameter) of each actively growing fungal straingrown on 2% MEA ground under sterile conditions in a 1.5 mL tube with1.0 mL of sterile water. Suspensions without fungal material were usedfor controls. Healthy foliage of 4-5 branches of each of the three planthosts were surface sterilized, allowed to air dry and immersed fullyinto fungal suspension (one tube per branch tip, 2 replicatetubes/strain or control/plant) and Parafilmed in place. Tubes wereremoved after 24 hours and branches were then bagged for 24 hours tomaintain high humidity. Viability of fungal inoculums were verified fromfungal suspensions directly applied to plant tissue. After two weeks,inoculated foliage was collected, cut into 10 2-mm² segments, surfacesterilized then plated together on 2% MEA and incubated at roomtemperature. The numbers of fungal and bacterial emergences wererecorded and identified based on morphology and molecular analysis.

Effect of EHB on Growth Rate Measurements.

Luteibacter reduces fungal growth at 25° C. for cultures grown on bothwater agar and malt extract agar but enhances growth for cultures grownon malt extract agar at 36° C. (EHB-Pestalotiopsis 9143 failed to growat this condition). MANOVA results are reported for conditions where EHBhas a significant growth effect on fungal growth rate. Error barsindicate standard error of the six replicates performed for allexperiments.

Media plates (100 mm in diameter) containing 15 ml of 2% MEA or wateragar were inoculated with a 6-mm pug of actively growing mycellium.Plates were incubated either at ˜25° C. or 36° C. (6 replicateplates/treatment) in the dark and fungal colony diameter was measured ontwo axes and averaged every two days for 18 days. Plates showingbacterial emergence as well as days where the colony reached the plateedge were excluded from MANOVA analysis.

EHB Influence Plant Infection by Fungal Endophytes.

Of the isolates that maintained EHB infection during plant inoculations,endophyte Microdiplodia 9145/Luteibacter was found after introductioninto C. arizonica. Microdiplodia 9140/Rhizobium was recovered from allthree plant species. However no isolates were recovered from plantmaterial inoculated with Microdiplodia 9140 (EHB−) or from thewater-only control. All source inocula were confirmed to be viable.

Non-limiting examples of endophytic fungi and endohyphal bacteria thatmay be used to effect the production of ligninase and/or cellulase, orgrowth at particular temperatures or on various types of nutrient media,are as shown in Table 1 below.

TABLE 1 Fungal Water Malt Water Malt endophyte Bacterium agar agar agaragar Lignin Cellulose Cellulase (EF) (EB) 25° C. 25° C. 36° C. 36° C.medium medium activity 9140- Pantoea ns − > + ns − > + ns ns nsMicrodiplodia sp. 9145- Erwinia + > − ns ns ns ns ns ns Microdiplodiasp. 9145- Luteibacter − > + − > + − > + + > − ns ns + > − Microdiplodiasp. 1 9055- Sphingomonas − > + − > + − > + − > + − > + ns + > −Alternaria sp. 9128- Burkholderia − > + ns − > + ns ns ns + > −Cladosporium sp. 9143- Luteibacter ns ns + > − ns ns + > − + > −Pestalotiopsis sp. 2

Table 1 shows context-dependence in the outcomes of EB-EF interactions(in the rows) and meaningful phenotypic variation among symbioticpartners (in the columns). “EB” refers to “endohyphal bacteria”, and“EF” refers to “endophytic fungi”. “+>−” indicates a statisticallysignificant enhancement of growth or enzyme activity when the funguscontains the EHB (+) relative to the fungus without the EHB (−). “−>+”indicates a statistically significant enhancement of growth or enzymeactivity when the fungus lacks the EHB (−) relative to the fungus thatcontains the EHB Not all of the combinations of fungi and bacteria shownin Table 1 result in a change cellulase activity.

Table 1 shows the significance and directionality of repeated-measuresANOVA assessing growth of clones in vitro over 14 days on water agar(low nutrient), malt extract agar (high nutrient), lignin medium(indulin as sole carbon source), and cellulose medium(carboxymethylcellulose as carbon source). Thermotolerance was assessedon two media at 36° C.

Experimental Data Regarding Ligninase Activity.

Methods.

We prepared eight fungal inocula: endophyte Pestalotiopsis 9143harboring its native endosymbiont, Luteibacter 9143; endophyteMicrodioplodia 9145 harboring its native endosymbiont, Luteibacter 9145;each endophyte growing axenically, after removal of bacterialendosymbionts via antibiotic treatments; each endophyte harboring itsnative endosymbiont after curing and resynthesis; and each endophyteharboring a novel endosymbiont (i.e., Pestalotiopsis 9143 withLuteibacter 9145, and Microdiplodia 9145 with Luteibacter 9143). We alsoprepared two bacterial inocula: Luteibacter 9143 and Luteibacter 9145growing axenically. The axenic bacteria did not grow on the indulinmedium and are excluded from analyses. For each inoculum (eight fungal,two bacterial), we inoculated six Petri dishes containing equal volumesof 2% water agar amended with 0.05% indulin. Inoculum consisted of a 6mm plug of actively growing mycelium, or a 3-day colony of bacteria.Inoculated plates were incubated at 22° C. for 10 days (fungus) or 3days (bacterium), then flooded with a 1.0% w/v FeCl₃ and K₃ [Fe(CN)]₆solution, incubated at room temperature for 40 min, and washed severaltimes with sterile water (Gazis et al., 2012). Colony diameter was usedas a proxy for growth rate. Clearing of the medium was noted as evidenceof ligninase activity. Colony diameter and zone of clearing werequantified by averaging the diameter of the colony or clearing acrosstwo axes.

Results

1. Inoculation of Fungi by EHB can Increase Fungal Growth Rate onIndulin Medium. The Increased Growth in the Inoculated Strains May beDue to an Increased Bacterial Titer Compared to Naturally InfectedFungal Strains.

The colony diameters are close enough to normally-distributed to arguefor using parametric statistics. These data appear to be grouped bytrial for the associations where we had experimental replication. Whenanalyzed together in a mixed-effects model, there was a significanteffect of bacterial treatment (fixed effect) on fungal growth whenaccounting for variation in trial (random effect) for bothPestalotiopsis 9143 and Microdiplodia 9145.

The following results are reported for trial 2, because this trialincluded results from the full sampling design.

1A. Growth of Pestalotiopsis on indulin medium was influenced bybacterial treatment (F_(3,19)=45.2367, p<0.00013). Colony diameter whenPestalotiopsis harbored non-native Luteibacter 9145 or when reassociatednative Luteibacter 9143 was significantly larger than Pestalotiopsisnaturally infected with Luteibacter 9143 or the bacteria-free fungalclone (Student's t tests with Bonferroni correction, adjustedalpha=0.005).

1B. Growth of Microdiplodia on indulin medium was influenced bybacterial treatment (F_(3,20)=8.2273, p=0.0009). Growth of Microdiplodiaon cellulose medium differed significantly as a function of bacterialstatus (Student's t tests and the Bonferroni adjusted alpha levels of0.005 per test). Colony diameter when Microdiplodia harbored non-nativeLuteibacter 9143 or when reassociated native Luteibacter 9145 wassignificantly larger than Microdiplodia naturally infected withLuteibacter 9145 or the bacteria-free fungal clone (Student's t testswith Bonferroni correction, adjusted alpha=0.005).

1C. For both fungal strains, the growth rates of the strains inoculatednative or non-native bacteria grew significantly better than thebacteria-free or naturally infected clones.

2. Ligninase activity (presence/absence of clearing outside or under thefungal colony) can be inhibited by presence of EHB (Microdiplodia) orEHB genotype (Pestalotiopsis).

2A. Within colony ligninase activity of both fungal strains differedqualitatively by infection status. Activity was observed in thebacteria-free clone of Microdiplodia, but not when the fungus harboredbacteria. Activity was also observed in the bacteria-free clone ofPestalotiopsis and when the fungus harbored Luteibacter 9143 but notLuteibacter 9145. Activity was consistent for all 6 replicates/trial anddid not differ by trial where there was trial replication.

3. EHB Presence (Microdiplodia) or Genotype (Pestalotiopsis) canDecrease Ligninase Activity Outside the Growing Edge of the FungalHost's Colony.

Zone of clearing scaled by colony diameter deviated significantly from anormal distribution, such that nonparametric statistics were used.

There is no trial effect of ligninase activity by bacterial treatment ineither Pestalotiopsis or Microdiplodia when activity by bacterialtreatment was compared with Mann-Whitney tests. Ligninase activityappears robust to fungal growth rate where we observed a difference ingrowth rate between the two trials.

The following results are reported for trial 2, since this trialincluded results from the full sampling design.

3A. Ligninase activity beyond the growing edge of Pestalotiopsiscolonies was influenced by bacterial treatment (χ²=11.8774, df=3,p=0.0078). Activity was greater when Pestalotiopsis harbored nativeLuteibacter 9143 (naturally infected) than when it was free frombacteria or when it harbored Lutiebacter 9145 for where there was noclearing beyond the colony edge. Means were compared with Mann-Whitneytests and the Bonferroni adjusted alpha levels of 0.005 per test.

3B. Ligninase activity beyond the growing edge of Microdiplodia colonieswas influenced by bacterial treatment (Ψ²=22.3952, df=3, p<0.0001).Ligninase activity outside Microdiplodia colonies was significantlygreater and was only observed in the bacteria-free clone. Means werecompared with Mann-Whitney tests and the Bonferroni adjusted alphalevels of 0.005 per test.

Experimental Data Regarding Cellulase Activity.

Methods.

We prepared eight fungal inocula: endophyte Pestalotiopsis 9143harboring its native endosymbiont, Luteibacter 9143; endophyteMicrodioplodia 9145 harboring its native endosymbiont, Luteibacter 9145;each endophyte growing axenically, after removal of bacterialendosymbionts via antibiotic treatments; each endophyte harboring itsnative endosymbiont after curing and resynthesis; and each endophyteharboring a novel endosymbiont (i.e., Pestalotiopsis 9143 withLuteibacter 9145, and Microdiplodia 9145 with Luteibacter 9143). We alsoprepared two bacterial inocula: Luteibacter 9143 and Luteibacter 9145growing axenically. For each inoculum (eight fungal, two bacterial), weinoculated six Petri dishes containing equal volumes of 2% malt extractagar amended with 0.5% carboxymethylcellulose. Inoculum consisted of a 6mm plug of actively growing mycelium, or a 3-day colony of bacteria.Inoculated plates were incubated at 22° C. for 10 days (fungus) or 3days (bacterium), then flooded with 0.2% w/v Congo red solution,incubated at room temperature for 40 min, and washed several times with1M NaCl (Gazis et al., 2012). Colony diameter was used as a proxy forgrowth rate. Clearing of the medium was noted as evidence of cellulaseactivity. Colony diameter and zone of clearing were quantified byaveraging the diameter of the colony or clearing across two axes.

Results

1. EHB Genotype can Influence Fungal Growth Rate on Cellulose Medium(Proxy=Colony Diameter).

The colony diameters are close enough to normally-distributed to arguefor using parametric stats. These data appear to be grouped by trial forthe associations where we had experimental replication. When analyzedtogether in a mixed-effects model, there was a significant effect ofbacterial treatment (fixed effect) on fungal growth when accounting forvariation in trial (random effect) for both Pestalotiopsis 9143 andMicrodiplodia 9145.

The following results are reported for trial 2, because this trialincluded results from the full sampling design.

1A. Growth of Pestalotiopsis on cellulose medium was influenced bybacterial treatment (F_(3,20)=6.0135, p=0.0043). Colony diameter whenPestalotiopsis harbored the non-native Luteibacter 9145 wassignificantly smaller than when Pestalotiopsis harbored Luteibacter 9143or had no bacterium (Student's t tests with Bonferroni correction,adjusted alpha=0.005). Colony diameter was similar when Pestalotopsisharbored Luteibacter 9143 or no bacterium. Colony diameter wassignificantly reduced when Pestalotiopsis harbored the non-nativeLuteibacter 9145.

1B. Growth of Microdiplodia on cellulose medium was influenced bybacterial treatment (F_(3,20)=49.7930, p<0.0001). Growth ofMicrodiplodia on cellulose medium differed significantly as a functionof bacterial status (Student's t tests and the Bonferroni adjusted alphalevels of 0.005 per test). Colony diameter when Microdiplodia harboredthe non-native Luteibacter 9143 was significantly greater than whenMicrodiplodia harbored Luteibacter 9145 or had no bacterium.

1C. For both fungal strains, only the growth rate of the straininoculated with the non-native bacterium significantly differed from thebacterium-free clone. In each case growth rate did not differ for fungiin the presence of their native bacterium vs. when growing axenically(i.e., with no bacterium). Growth of Pestalotiopsis 9143 wassignificantly reduced relative to axenic strains by the presence of thenon-native bacterium 9145. Growth of Microdiplodia with 9143 wassignificantly slower than that of Pestalotiopsis 9143 when the samebacterial strain was present in each. Growth of Microdiplodia 9145 withbacterium 9143 was significantly faster than growth of Microdiplodiaalone. In both cases, strains that harbored Luteibacter 9143 tended togrow more than strains that harbored Luteibacter 9145.

2. Cellulase Activity (Presence/Absence of Clearing Outside or Under theColony) is Only Observed in the Presence of EHB.

2A. Within colony cellulase activity of both fungal strains differedqualitatively by infection status. Activity was observed whenMicrodiplodia and Pestalotiopsis harbored Luteibacter 9143 orLuteibacter 9145. No activity was observed in the bacteria-free clones.Activity was consistent for all 6 replicates/trial and did not differ bytrial where there was trial replication.

3. EHB Genotype can Increase Cellulase Activity Outside the Growing Edgeof the Fungal Host's Colony. Without Bacteria, No Activity Outside theColony was Present in Either Fungal Strain.

Zone of clearing scaled by colony diameter deviated significantly from anormal distribution, such that nonparametric statistics were used. Thereis no trial effect of cellulase activity by bacterial treatment ineither Pestalotiopsis or Microdiplodia when activity by bacterialtreatment was compared with Mann-Whitney tests. Cellulase activityappears robust to fungal growth rate where we observed a difference ingrowth rate between the two trials. As shown in, zone of celluloseclearing scaled by fungal colony diameter of both trials grouped byfungal strain (Pestalotiopsis 9143 or Microdiplodia 9145, bacterialstrain (Luteibacter 9143 or 9145) and type of association (+=naturallyinfected, R=re-associated). Data points from trial 2 are gray and datafrom trial 1 are black.

The following results are reported for trial 2, since this trialincluded results from the full sampling design.

3A. Cellulase activity beyond the growing edge of Pestalotiopsiscolonies was influenced by bacterial treatment (χ²=9.774, df=3,p=0.0206). Activity was greater when Pestalotiopsis harbored nativeLuteibacter 9143 than when it was free from bacteria or when it harboredLuteibacter 9145 for where there was no clearing beyond the colony edgewhen compared with Mann-Whitney tests and the Bonferroni adjusted alphalevels of 0.005 per test.

3B. Cellulase activity beyond the growing edge of Microdiplodia colonieswas influenced by bacterial treatment (χ²=13.3295, df=3, p=0.004).Cellulase activity outside Microdiplodia colonies differed significantlyas a function of infection status when compared with Mann-Whitney testsand the Bonferroni adjusted alpha levels of 0.005 per test. Activity wassignificantly greater when Microdiplodia harbored Luteibacter 9143 orLuteibacter 9145 compared to the bacteria-free clone for which there wasno activity outside the colony. Conclusions. Cellulase production isrobust to variation in fungal growth rates. Infection by a non-nativebacterium has the greatest influence on fungal growth rate, and thedirection of change is dependent on the type of association. Cellulaseactivity was only observed in fungal strains that harbored bacteria andthe axenic bacteria. The degree of activity may be influenced by thebacterial genotype in Pestalotiopsis. In all responses examined fornative associations, naturally infected fungal phenotypes were recoveredin the re-associated strains suggesting bacterial presence isresponsible for altering fungal phenotypes in natural associations.

Experimental Data Illustrating Transfer of EHB Between Fungi fromDifferent Classes of Ascomycota.

The following describes methods for successfully reintroducing(re-synthesizing or re-associating) EHB into axenic fungal mycelia, andillustrates that the identity of the fungal host and culture conditionscan define the establishment of these widespread and importantsymbioses. We first establish in vitro conditions favoringreintroduction of two strains of EHB (Luteibacter spp.) into axenicstrains of their original fungal hosts, focusing on two fungi originallyisolated from healthy plant tissue as endophytes: Microdiplodia sp.(Dothideomycetes) and Pestalotiopsis sp. (Sordariomycetes). We show thatreintroduction was successful for Microdiplodia/Luteibacter on potatodextrose agar and water agar, but was only successful forPestalotiopsis/Luteibacter on water agar. We demonstrate that these EHBcan be introduced to a novel fungal host under the same conditions,successfully transferring EHB between fungi representing differentclasses of Ascomycota. Finally, we manipulate conditions to optimizereintroduction in a focal EHB/fungal association, altering the nutrientcontent for the co-culture medium, the mycelium:bacteria ratio inco-culture, the age of the bacterial culture at the time ofco-culturing, treatment of the axenic cultures prior to co-culturing,and the nutrient content of the solid medium onto which the co-culturewas plated. We show that EHB infections were initiated and maintainedmore often under low-nutrient culture conditions and when EHB and fungalhyphae were washed with MgCl₂ prior to re-association.

Here we examine methods for successfully reintroducing EHB into axenicfungal mycelia, with a focus on two species representing distantlyrelated clades of Ascomycota that were originally isolated as foliarendophytes from a woody plant. We first establish in vitro conditionsfavoring reintroduction of two different strains of axenic EHB(Luteibacter sp., Gammaproteobacteria) into axenic strains of theiroriginal fungal hosts. We then demonstrate that these EHB can beintroduced to novel fungal hosts under the same conditions, successfullytransferring EHB between members of the Dothideomycetes andSordariomycetes. Finally, we manipulate conditions to optimizereintroduction in a focal EHB/fungal association, examining theimportance of the nutrient content for the co-culture medium, themycelium:bacteria ratio in co-culture, the age of the bacterial cultureat the time of co-culturing, treatment of the axenic cultures prior toco-culturing, and the nutrient content of the solid medium onto whichthe co-culture was plated.

Our study provides a new suite of methods for experimental assessment ofthe effects of EHB on fungal phenotypes, and suggests that both thefungal host and culture conditions can influence the establishment ofthese widespread and important symbioses. By understanding how thesesymbioses are initiated and maintained, we can gain new insights intothe cryptic ecological interactions that shape ubiquitous plant-fungalassociations. In turn, by manipulating EHB/fungal interactions in newways, we can potentially influence fungal phenotypes for diverse humanapplications.

Materials and Methods

As part of a previous study, endophytes were isolated from healthy,surface-sterilized foliage of Platycladus orientalis (Cupressaceae) inDurham, N.C. (Hoffman and Arnold 2010). This collection includedPestalotiopsis sp. 9143 (Amphisphaeriaceae, Xylariales, Sordariomycetes)with its naturally occurring bacterial symbiont, Luteibacter sp. 9143;and Microdiplodia sp. 9145 (Botryosphaeriaceae, Botryosphaeriales,Dothideomycetes) with its naturally occurring symbiont, Luteibacter sp.9145. Although the fungi represent distinct classes of Ascomycota, thebacteria are closely related: their 16S rRNA sequences are 100%identical, and whole genome sequences are nearly invariant (Baltrus etal. in prep). Both associations are accessioned as living cultures atthe Robert L. Gilbertson Mycological Herbarium at the University ofArizona (accessions MYCO-ARIZ 9143 and 9145).

Preparation of Axenic Cultures

Each fungal strain was cured of EHB by cultivation on 2% malt extractagar (MEA) amended with four antibiotics: ampicillin (100 μg/ml),kanamycin (50 μg/ml), tetracycline (10 μg/ml), and ciprofloxacin (40μg/ml) (see Fisher et al. 1996, Rodrigues 1994, Lodge et al. 1996,Gamboa and Bayman 2001, Hoffman and Arnold 2010). We confirmed thatfungal cultures were free of EHB using the methods described below.

EHB were isolated from naturally infected fungal cultures on 2% MEA (seeHoffman and Arnold 2010) that were incubated for 72 h at 36° C. At thistemperature, bacteria emerged from hyphae and were isolated by streakingonto Luria broth (LB) agar (Bertani 1952). Unless otherwise stated,axenic fungal strains were maintained on 2% MEA at 25° C. and axenicbacterial strains were maintained in LB at 25° C.

Reintroduction of EHB into Axenic Host Fungi

We first reintroduced EHB into axenic strains of their native hostfungus (i.e., Luteibacter sp. 9143 in Pestalotiopsis sp. 9143, andLuteibacter sp. 9145 in Microdiplodia sp. 9145). Prior to reassociation,the fungal and bacterial strains were prepared as follows.

For each axenic fungus, a plug of mycelium (1.25 cm diameter) wascollected under sterile conditions from inside the edge of an activelygrowing colony on 1× potato dextrose agar (PDA, 2.4%). Each plug wasseparately blended in three 5 sec, high-speed pulses in a sterileblender (Waring 51BL31) in 100 mL 1× potato dextrose broth (PDB), andthen transferred to a sterile flask and incubated on a rotary shaker at27° C. and 100 rpm for 7 d. Mycelium was collected via vacuum filtrationonto 8 μm Whatman filter papers, washed twice with sterile 10 mM MgCl₂,removed from the filter papers with forceps under sterile conditions,resuspended in 100 mL of 1×PDB, and blended as before. Removing ordiluting the supernatant from liquid axenic cultures and washingcultures in a neutral buffer prior to co-culturing is common inmicrobial competition studies (Lenski et al. 1991, Jankowska et al.2008) to maintain an osmotic balance between the internal and externalenvironment of the cells.

Bacterial cultures were first grown on LB agar and then inoculated into5 mL of LB and incubated on a rotary shaker at 36° C. and 200 rpm for 3d. Cultures then were centrifuged at 300 rcf for 3 min and thesupernatant was discarded. The pelleted cells were washed twice with 4mL of sterile 10 mM MgCl₂ and resuspended in 4 mL of 1×PDB.

Before reassociation, fungal and bacterial suspensions were evaluatedwith a spectrophotometer (OD 600), normalized with respect to axenicbacterial inocula (5:1 mycelium:bacteria), and added to 50 mL of 1×PDB.Because we observed no selectable phenotype for the reassociated strainswhen grown on standard fungal media, we chose this ratio because athigher concentrations of bacteria, we often observed bacteria growingepihyphally on fungal cultures. The resulting mixture was cultured for 7d at 27° C. in full darkness, with agitation on a rotary shaker at 100rpm.

Each co-culture was prepared twice. After incubation, 20 μl of eachco-culture was transferred to six Petri dishes containing nutrientmedia: three plates contained 1×PDA and three contained water agar.Plates were incubated at 27° C. for 14 d. Bacterial infection status wasverified as described below. The success of establishing symbioses wasconsistent across all replicates on each medium for each EHB/fungalassociation. After successful infection, fungi were subcultured threetimes on 2% MEA to confirm the stability of the association.

We also attempted to reassociate two additional EHB/foliar fungalassociations described in Hoffman and Arnold (2010): Phyllosticta sp.9135 (Dothideomycetes) with its Luteibacter sp., and Botryosphaeria sp.9133 (Dothideomycetes) with its Luteibacter sp. Neither of theseassociations was reassociated successfully under the conditions above,and thus these trials are excluded from the results.

Cross-Inoculation

Next, EHB were introduced into axenic strains of the alternate fungalhosts (i.e., Luteibacter sp. 9145 in Pestalotiopsis sp. 9143, andLuteibacter sp. 9143 in Microdiplodia sp. 9145). All methods followedthose described above.

Manipulation of Resynthesis Conditions

We focused on the association of Pestalotiopsis sp. 9143 withLuteibacter sp. 9143 to further examine culture conditions under whichEHB could be introduced. Using conditions that permitted successfulreassocation as a baseline protocol (above), we altered the nutrientcontent for the co-culture medium (1×, 0.1×, 0.01×, 0.001×, and0.0001×PDB) and the mycelium:bacteria ratio in the co-culture (10:1,7:1, and 5:1). We also examined the role of the age of the bacterialculture at the time of co-culturing (1 day old vs. 3 days old), whetheraxenic cultures were washed with MgCl₂ before co-culturing, and thenutrient content of the solid medium onto which the co-culture wasplated (water agar vs. 1×PDA). For this experiment, we adjusted theco-culture volume to 5 mL to include more replicates. Co-cultures werethen incubated for 3 d in culture tubes. Each treatment was replicatedtwice and one fungal colony from each replicate was screened for EHB.The role of resynthesis conditions was quantified using nominal logisticanalysis, with success of association as the response variable (yes, no)and the above treatments as explanatory variables. Successfulassociation (i.e., ‘yes’) was defined by detection of the bacteriumusing molecular analysis and confirmation that the bacteria were viable,endohyphal, and occurring within viable fungal hyphae based onvisualization, as described below.

Molecular Analysis and Identification of EHB

Genomic DNA was extracted directly from fresh fungal mycelium collectedfrom inside the growing edge of a fungal colony using a modifiedprotocol from the Extract-N-Amp tissue PCR kit (Sigma-Aldrich). GenomicDNA was screened for the presence of bacteria by 16S rRNA PCR usingRedTaq (Sigma) with primers 27F/1492 (Lane 1991). PCR conditionsfollowed Hoffman and Arnold (2010) with the following amendments: 50° C.annealing temperature and 40 cycles.

Positive 16S rRNA PCR products were cleaned using ExoSAP-IT (Affymetrix)and Sanger-sequenced bidirectionally at the University of ArizonaGenetics Core. Sequences were assembled automatically, bases called, andquality scores assigned by phred (Ewing and Green 1998) and phrap (Ewinget al. 1998) with orchestration by Mesquite v. 1.06 (Maddison andMaddison 2011). Consensus sequences were edited manually in Sequencher5.1 (Gene Codes Corporation) and compared against sequences obtainedfrom the same specimens by Hoffman and Arnold (2010).

In all cases, sequences of bacteria obtained here were 100% identical tothose reported previously from these cultures (Hoffman and Arnold 2010).Sequences also were BLASTed against GenBank (BLASTn, highly similarsequences; Altschul et al. 1990, Benson et al. 2014). Taxonomicplacement within Luteibacter, validated previously by phylogeneticanalysis (Hoffman et al. 2013), was confirmed using a ≥99% match overthe full sequence length. As needed, the same methods were used toconfirm the identity of EHB growing axenically.

We did not identify any additional EHB or free-living bacteria in thefungal cultures used in this study. Negative PCR products (i.e., thosefor which no bands were evident after 16S rRNA PCR) were cloned(Agilent, StrataClone) following the manufacturer's instructions forreactions using half volumes. No positive clones were recovered fromfungi after antibiotic treatment or from negative controls.

Visualization Methods

Molecular analyses were coupled with visual assessments to confirm thatEHB were viable and were present within living fungal hyphae. Visualassessments consisted of microscopy and staining using the LIVE/DEADBacLight Bacterial Viability Kit (Invitrogen) following Hoffman andArnold (2010).

To prepare fungal samples for visualization, fresh hyphae were scrapedfrom the surface of the growing edge of each fungal colony on 2% MEA.Axenic bacteria were prepared by scraping a single colony from LB agar.Hyphae or bacterial cells were placed on a glass slide with 15 μl of1:1:18 LIVE/DEAD stain (component A: component B: diH₂O), covered with acoverslip, and incubated in darkness for 20 min. Sterile distilled waterthen was pulled through the slide mount with blotting paper. Slides weresealed with clear acrylic nail polish, which was allowed to dry beforeviewing. A Leica 4000 MB compound microscope with a 100-W mercury arclamp was used for fluorescent imaging at room temperature with a ChromaTechnology 35002 filter set (480-nm excitation/520-nm emission) and100×APO oil objective. Three replicate slides were prepared per fungalculture, and in all cases, replicates from the same material wereconsistent.

Results

Both Pestalotiopsis sp. 9143 and Microdiplodia sp. 9145 were viable on1×PDA and water agar in the presence and absence of EHB. Each strain ofLuteibacter was viable on LB agar and in LB under the conditionsdescribed above, and could be isolated reliably following heat treatmentof infected mycelia. EHB could be detected reliably from the originalcultures using the above molecular and visualization methods, and wereconfirmed to be absent following antibiotic treatment.

Reintroduction of EHB

EHB were successfully reintroduced to their original host strains afterthose fungi were treated with antibiotics. In each case the bacteriawere confirmed to be endohyphal and viable. Reinfected strains resembledthe naturally infected strains with regard to hyphal morphology.

Reintroduction of Luteibacter sp. 9145 into Microdiplodia sp. 9145 wassuccessful when the co-culture was plated on PDA or water agar. However,reintroduction of Luteibacter sp. 9143 into Pestalotiopsis sp. 9143 wasonly successful on water agar. In each case, reinfected fungal strainsmaintained these associations through at least three subculturing eventson 2% MEA. Reisolation and confirmation of bacterial identity followingreintroduction is now being completed.

Cross-Inoculation of EHB

Luteibacter sp. 9143 was successfully introduced into Microdiplodia sp.9145 when the co-culture was plated on either PDA or water agar.Luteibacter sp. 9145 was successfully introduced into Pestalotiopsis sp.9143 when the co-culture was plated on water agar. In each case,bacteria were confirmed to be endohyphal and viable and were maintainedin their novel fungal hosts through at least three subculturing eventson 2% MEA. Reinfected strains resembled the naturally infected strainswith regard to hyphal morphology and the density of bacterial cells.Reisolation and confirmation of bacterial identity followingreintroduction is now being completed.

Manipulation of Resynthesis Conditions

We examined the effects of particular culture conditions onreintroduction of EHB by focusing on the association betweenPestalotiopsis sp. 9143 and Luteibacter sp. 9143. A total of 120 trialswas conducted, each representing a bacterial culture of a given age (1day old or 3 day old); washing of axenic cultures with MgCl₂ or not;various concentrations of the medium in which the co-culture was grown(1×, 0.1×, 0.01×, 0.001×, and 0.0001×PDB); various mycelium:bacteriaratios in the co-culture (10:1, 7:1, and 5:1); and cultivation of theco-culture on 1×PDA or water agar. All treatment combinations and theiroutcomes are shown in Table 2.

Nominal logistic analysis revealed that when all culture conditions wereconsidered, those most relevant for successful resynthesis were (1)whether the axenic cultures were washed in MgCl₂, (2) the concentrationof PDB in which the co-culture was grown, and (3) the solid medium onwhich the co-culture was plated. Culture age and the ratio ofmycelium:bacteria were less important in the overall analysis.

In vitro reassociation of Pestalotiopsis sp. 9143 and Luteibacter sp.9143 always failed when axenic cultures were not washed with MgCl₂ andwhen co-cultures were cultivated in 0.0001×PDB, and failed in 29 of 30trials when the co-cultures were plated on 1×PDA. This table showsqualitative outcomes of resynthesis attempts when cultures were washedwith MgCl₂, PDB concentrations were >0.0001×, and co-cultures wereplated on water agar for resynthesis attempts started with 1- and 3-dayold bacterial cultures. Nominal regression of this reduced data set didnot reveal significant differences among the suites of treatments listedhere; however, we note that resynthesis was always successful when0.01×PDB was used as the co-culture medium, whereas success was morevariable on other concentrations of PDB.

Examination of the data revealed that resynthesis failed in all 60trials in which axenic cultures were not washed with MgCl₂. We thusexcluded those 60 trials from further analysis. Among the remaining 60trials, resynthesis was more often successful when the co-culture wasplated on water agar rather than on 1×PDA: only one of 30 resynthesisattempts using PDA was successful (3 d old bacterial culture, 0.01×PDB,7:1 mycelium:bacteria ratio, whereas 18 of 30 resynthesis attempts weresuccessful using water agar. We thus excluded the 30 trials on PDA fromfurther analysis. These findings are consistent with the results withour initial assessment of the influence of the culture medium on thesuccess of reintroducing Luteibacter 9143 into Pestalotiopsis 9143.

Among the remaining 30 trials, all resynthesis attempts wereunsuccessful when the co-culture was grown in 0.0001×PDB, but someresynthesis attempts were successful on each of the remainingconcentrations of PDB. We thus excluded the trials on 0.0001×PDB fromfurther analysis.

We then analyzed the remaining data set to more precisely evaluate theimportance of the age of the bacterial culture, the concentration of PDB(1×, 0.1×, 0.01×, 0.001×), and the ratio of the mycelium:bacteria inco-culture establishment. Nominal regression of this reduced data setdid not reveal significant differences among the suites of treatmentslisted here (simplified whole model, chi-square=8.57, df=6, P=0.1993; nosignificant effects of any factor: P=0.2659, P=0.1009, and P=0.04009,respectively). However, resynthesis was always successful when 0.01×PDBwas used as the co-culture medium, whereas success was more variable onother concentrations of PDB.

The present aforementioned study demonstrates resynthesis in anassociation with foliar fungal endophytes in the species-richAscomycota, which includes the vast majority of endophytic andplant-pathogenic fungi of relevance to agriculture and natural systems.This also demonstrates resynthesis involving Gammaproteobacteria, whichare common in foliar endophytes studied to date, and can have profoundeffects on fungal. We demonstrate resynthesis for two strains of EHB andtwo distantly related fungi, and demonstrate that EHB can be movedbetween fungal strains.

The present study focused on two closely related bacteria. We believethat EHB of foliar fungi that are horizontally acquired are not hostspecific, although an important caveat is that all work to date hasrelied only on 16S rRNA sequences to differentiate bacterial strains.Such an approach alone would indicate that Luteibacter sp. 9143 and 9145are of the same species; their 16S rRNA sequences are 100% identical,and whole genome sequences are nearly invariant.

Facultative Symbioses

We found that the EHB association was facultative in these foliar fungi.Both fungi and bacteria could be grown axenically on standard media.

Nutrient Conditions

Reassociation was successful for both Pestalotiopsis sp.9143/Luteibacter sp. 9143 and Microdiplodia sp. 9145/Luteibacter sp.9145 on low-nutrient media (water agar). When grown on water agar,hyphae of both fungal species were sparse, transparent, and thin; incontrast, both strains had robust, dense, and pigmented or opaque hyphaeon PDA. Resynthesis of Microdiplodia sp. 9145 with Luteibacter 9145 alsowas successful on high-nutrient media. Cross-infection of both fungalhosts with novel EHB displayed the same patterns as those observed withtheir native EHB.

We found that available nutrients in the culture medium stronglyinfluenced the success of establishing EHB symbioses. Additionally,resynthesis was only successful when both axenic cultures were washedwith MgCl₂, implying the presence of biological inhibitors that wereremoved upon washing. This is not surprising as many microbes will exudeantimicrobial compounds that can interfere with the functions of closelyrelated species or organisms in different kingdoms (Brian and Hemming1947, Be'er et al. 2008). This competition is especially common betweenmicrobial endophytes (Strobel et al. 2004).

Manipulation of Culture Conditions

Our results suggest that when cultures are washed with MgCl₂, PDBconcentrations are ca. 0.001× or greater, and co-cultures are plated onwater agar, resynthesis of Pestalotiopsis sp. 9143/Luteibacter sp. 9143can be achieved at various ratios of mycelium:bacteria (5:1, 7:1, 10:1),various concentrations of PDB (1×, 0.1×0.01×, 0.001×), and with 1 or 3 dold bacterial cultures. Qualitative examination of the results indicatesthat the most consistent success was obtained using 0.01×PDB, but thesedifferent suites of conditions did not differ statistically.

When comparing resynthesis success with 1 vs. 3 day old bacterialcultures, we observed the trend that trials started with the 3 day oldbacterial culture reestablished symbiosis when the mycelium:bacteriaratio was lower and when the nutrient content of the co-culturing mediumwas higher than trials started with the 1 dayold culture. This could bedue to a difference in the growth stage of the bacterium at the time ofco-culturing. We also noted that when co-cultures started with the 1 dayold bacterial culture were plated on 1×PDA after cultivation in 1×PDB,viable bacteria often were observed living outside of fungal cells. Assuch, resynthesis per se could not be verified. We therefore wereconservative in our analyses and treated these as unsuccessfulreintroductions.

Experimental Data Illustrating the Effects of Two Closely Related EHBsof the Genus Luteibacter on Substrate Use, Cellulase Activity, LigninaseActivity and Plant Tissue Degradation.

FIGS. 1-6 illustrate the results of various tests described below.

FIG. 1 shows results of in vitro mass-loss experiment on fresh andsenescent tissue of Juniperus deppeana and Cupressus arizonica as afunction of treatment with seven fungi with (+) and without (−) EHB. (A)fresh material of J. deppeana, (B) senescent material of J. deppeana,(C) fresh material of C. arizonica, and (D) senescent material of C.arizonica. Error bars represent standard error about the mean. Differentletters indicate significantly different means from post hoc tests.

FIG. 2 shows mean colony diameter of Pestalotiopsis sp. 9143,Microdiplodia sp. 9145, and axenic bacteria during cellulase assays as afunction of EHB (Luteibacter sp. 9143 or Luteibacter sp. 9145) and typeof association (+=naturally infected, R=re-associated, none=nobacterium/axenic). Error bars represent one standard error. Differentletters indicate significantly different means from post hoc tests.

FIG. 3 shows average colony diameter of Pestalotiopsis sp. 9143 orMicrodiplodia sp. 9145 on indulin medium as a function of bacterialstrain (Luteibacter sp. 9143 or Luteibacter sp. 9145) and type ofassociation (+=naturally infected, R=re-associated, none=nobacterium/axenic). Error bars represent one standard error. Differentletters indicate significantly different means from post hoc tests.

FIG. 4 shows results of in vitro mass-loss experiment for P. orientalisfoliage, including (A) fresh, green leaf material and (B) senescent leafmaterial treated with each of seven foliar fungi with (+) and without(−) EHB. Error bars represent standard error about the mean. Differentletters indicate significantly different means from post hoc tests.

FIG. 5 shows in vitro mass loss from foliage of P. orientalis as afunction of foliage state (fresh, senescent) and EHB status for eachfungus. (A) Pestalotiopsis sp. 9143, fresh material; (B) Microdiplodiasp. 9145, fresh material; (C) Pestalotiopsis sp. 9143, senescentmaterial; (D) Microdiplodia sp. 9145, senescent material. Type ofassociation: +=naturally infected, R=re-associated, none=no bacterium.Error bars represent standard error about the mean. Different lettersindicate significantly different means from post hoc tests.

FIG. 6 shows the relationship of mass loss vs. the visual score offungal growth, with the 95% confidence interval for the linear fitshaded along the best-fit line.

We manipulated two endophyte/EHB associations to examine enzyme activityand measure the resulting effects on plant tissue degradation. We foundthat the presence and identity of EHB significantly influenced fungalgrowth and cellulase and ligninase activity in a partnership-specificmanner. Relative to axenic controls, fungal cultures infected with EHBgrew significantly more rapidly on, and led to greater mass loss from,senescent tissue of their host plant species vs. confamilial plantspecies. However, EHB-infected and EHB-free strains did not differ intheir capacity to grow on or degrade fresh plant material or materialfrom related hosts.

We evaluated the effects of two closely related EHB in the genusLuteibacter (Gammaproteobacteria) on substrate use, cellulase activity,ligninase activity, and plant tissue degradation by two species offoliar endophytes in the Pezizomycotina (Pestalotiopsis sp. 9143,Sordariomycetes; Microdiplodia sp. 9145, Dothideomycetes). Specifically,we test the hypotheses that the presence and identity of EHB influenceendophyte growth on cellulose- or ligin-based media, the presence and/orextent of fungal cellulase or ligninase activity, and the capacity ofendophytes to degrade living and senescent foliage of their host speciesand related trees.

Materials and Methods

As part of a previous study, focal endophytes were isolated fromhealthy, surface-sterilized foliage of Platycladus orientalis(Cupressaceae) in Durham, N.C. (Hoffman and Arnold 2010): Pestalotiopsissp. 9143 with its naturally occurring bacterial symbiont, Luteibactersp. 9143, and Microdiplodia sp. 9145, with its naturally occurringbacterial symbiont, Luteibacter sp. 9145. The fungi are distantlyrelated members of the Ascomycota, representing distinct classes(Sordariomycetes and Dothideomycetes, respectively). The Luteibacterspecies are closely related; their 16S rRNA sequences are 100%identical, and whole genome sequences are nearly invariant (Baltrus etal. in prep). Luteibacter sp. 9143 has been shown to enhance productionof indole-3-acetic acid by Pestalotiopsis sp. 9143 (Hoffman et al.2013), but other aspects of their interactions, and functional aspectsof the association between Microdiploda sp. 9145 and Luteibacter sp.9145, have not been evaluated previously. Both associations areaccessioned as living cultures at the Robert L. Gilbertson MycologicalHerbarium at the University of Arizona (accessions MYCO-ARIZ 9143 and9145).

We prepared eight fungal inocula from these strains: endophytePestalotiopsis sp. 9143 with Luteibacter sp. 9143; endophyteMicrodioplodia sp. 9145 with Luteibacter sp. 9145; each endophytegrowing axenically after removal of EHB via antibiotic treatments (i.e.,cured strains; see below); each endophyte with its native EHB afterremoval and resynthesis of the association; and each endophyte afterremoval of EHB and cross-infection with a novel EHB (Pestalotiopsis sp.9143 with Luteibacter sp. 9145, and Microdiplodia sp. 9145 withLuteibacter sp. 9143). We also prepared two bacterial inocula:Luteibacter sp. 9143 and Luteibacter sp. 9145, each growing axenically.Unless otherwise stated, fungal strains were maintained on 2% maltextract agar (MEA) at 25° C. and bacterial strains were grown in Luriabroth (Bertani 1952) at 25° C.

To establish axenic fungal cultures, each endophyte was cured of its EHBby cultivation on 2% MEA amended with four antibiotics: ampicillin (100μg/ml), kanamycin (50 g/ml), tetracycline (10 μg/ml), and ciprofloxacin(40 μg/ml). To resynthesize native associations and to inoculateendophytes with novel EHB, each axenic fungus was blended individuallyin three, 5 sec, high-speed pulses in a sterile blender (Waring 51BL31).Fungal suspensions were quantified with a spectrophotometer (OD 600) andnormalized with respect to axenic bacterial inocula at a ratio of 5:1mycelium:bacteria. The resulting mixture was co-cultured in 1× potatodextrose broth for 7 days at 27° C. in full darkness, with agitation ona rotary shaker at 100 rpm. After incubation, each co-culture was platedon 2% water agar. Bacterial infection status was verified as describedbelow.

Molecular Analyses of EHB

Genomic DNA was extracted directly from fresh fungal mycelium collectedfrom inside the growing edge of a fungal colony using a modifiedprotocol from the Extract-N-Amp tissue PCR kit (Sigma-Aldrich). Myceliumwas ground with a sterile pestle prior to heat lysis. Genomic DNA wasscreened for the presence of bacteria by 16S rRNA PCR using RedTaq(Sigma) and primers 27F/1492 (Lane 1991). PCR conditions followedHoffman and Arnold (2010) with the following amendments: 50° C.annealing temperature and 40 cycles. Positive and negative controls wereused in every PCR.

Positive 16S rRNA PCR products were cleaned using ExoSAP-IT (Affymetrix)and Sanger-sequenced bidirectionally at the University of ArizonaGenetics Core. Sequences were assembled automatically, bases called, andquality scores assigned by phred (Ewing and Green 1998) and phrap (Ewinget al. 1998) with orchestration by Mesquite v. 1.06 (Maddison andMaddison 2011).

Consensus sequences were edited manually in Sequencher 5.1 (Gene CodesCorporation) and compared against the known sequences obtained from thesame specimens by (Hoffman and Arnold 2010). In all cases, sequenceswere 100% identical to those of the previously studied samples.Sequences also were queried against GenBank using BLASTn (Altschul etal. 1990, Benson et al. 2014). Taxonomic placement within Luteibacter,validated previously by phylogenetic analysis for 9143 (Hoffman et al.2013), was confirmed using a ≥99% match over the full sequence length.As needed, the same methods were used to confirm the identity of EHBgrowing axenically.

We did not identify any additional EHB or free-living bacteria in thefungal cultures used in this study. Negative PCR products (i.e., thosefor which no bands were evident after 16S rRNA PCR) were cloned(Agilent, StrataClone) following the manufacturer's instructions forreactions using half volumes. No positive clones were recovered fromfungi after antibiotic treatment or from negative controls.

LIVE/DEAD staining. Microscopy and staining using the LIVE/DEAD BacLightBacterial Viability Kit (Invitrogen) was used to confirm that EHB infungal mycelia that were positive in the 16S assay were not presentoutside of fungal hyphae (i.e., were not ectosymbiotic or extrahyphal)and were viable (Hoffman and Arnold 2010). A Leica 4000 MB compoundmicroscope with a 100-W mercury arc lamp was used for fluorescentimaging at room temperature with a Chroma Technology 35002 filter set(480-nm excitation/520-nm emission) and 100×APO oil objective.

To prepare samples for visualization, fresh hyphae were scraped from thesurface of the growing edge of each colony on 2% MEA. Hyphae were placedon a glass slide with 15 μl of 1:1:18 LIVE/DEAD stain (component A:component B: diH₂O), covered with a coverslip, and incubated in darknessfor 20 min. Sterile distilled water then was pulled through the slidemount with blotting paper. Slides were sealed with clear acrylic nailpolish, which was allowed to dry before viewing. Three replicate slideswere prepared per fungal culture, and in all cases, replicates from thesame material were consistent. The presence of viable EHB was defined bypositive PCR results and a lack of extrahyphal or ectosymbiotic bacteria(as determined by comparison with contaminated strains).

In Vitro Cellulase and Ligninase Activity Assays

For each of ten inocula (eight fungal, two bacterial), we inoculated sixPetri dishes containing equal volumes of 2% MEA amended with 0.5%carboxymethylcellulose (i.e., cellulose medium; cellulase assay) or 2%water agar amended with 0.05% indulin (i.e., lignin medium; ligninaseassay) following Gazis et al. (2012). Each fungal inoculum consisted ofa 6 mm plug of actively growing mycelium, which was placed on the assayplate with the mycelial surface in contact with the medium. Eachinoculum for axenic bacteria consisted of a transfer by sterile loopfrom a three-day-old colony on 2% MEA. Inoculated plates were incubatedat 22° C. in darkness for 10 day (fungal inocula) or three day (axenicbacterial inocula). Colony diameter was then was measured with a ruler.Measurements were taken on two perpendicular axes that intersected atthe colony center, and were averaged to yield the final diameter value.

To evaluate clearing of the medium due to enzyme activity (i.e., toquantify enzyme activity), hyphae were scraped from the surface of theplate with a rubber policeman and sterile water. Plates then wereflooded with a 0.2% w/v Congo red solution (cellulase assay) or a 1.0%w/v FeCl₃ and K₃ [Fe(CN)]₆ solution (ligninase assay), incubated at roomtemperature for 40 min, and washed several times with 1M NaCl (Gazis etal. 2012).

Clearing of the medium either beneath colonies or beyond the growingedges provided evidence of enzyme activity. If present, the extent ofactivity was evaluated by measuring the zone of clearing beyond thecolony edge. The zone of clearing was measured with a ruler on twoperpendicular axes as above.

The presence and extent of enzyme activity was evaluated twice for eachfungus with its native bacterial associate and when grown axenically.These assays were conducted once for the resynthesized associations andnovel associations, and for the bacteria growing axenically.Presence/absence of activity was consistent across all replicates foreach inoculum in each trial, and did not differ by trial when trialswere replicated. Here we focus on the second trial because the firsttrial did not include all inoculum types, results of the first andsecond trials were qualitatively consistent, and because the first trialwas conducted at a lower temperature (22° C.), resulting in less growththan was observed in the second trial.

Statistical Analyses for Growth and Enzyme Assays

Colony diameter for each fungal inoculum type was compared within eachassay type to determine effects of EHB presence and identity on fungalgrowth and enzyme activity. Diameter values were normally distributedand were compared with ANOVA, followed by post-hoc Student's t-tests. ABonferroni-adjusted alpha level of 0.0083 was used for each post-hoctest. Colony diameters for axenic bacteria were obtained on cellulaseassay plates, but these bacteria did not grow on ligninase assay plates.Their diameter values on cellulase assay plates were normallydistributed and were compared (i.e., 9143 vs. 9145) using a t-test.

The extent of clearing for each fungal inoculum was scaled by colonydiameter prior to analysis. Values were then compared to determineeffects of EHB presence and identity. Because enzyme activity valueswere not normally distributed, means were compared using aKruskal-Wallis test, followed by post-hoc Mann-Whitney tests. ABonferroni-adjusted alpha level of 0.0083 was used for each post-hoctest. No clearing of the medium was observed beyond the colony edge forthe axenic bacteria on cellulase assay plates. All analyses were carriedout in JMP v. 11.0.0 (SAS Institute, Cary, N.C., USA).

In Vitro Mass-Loss Experiments

Fresh (i.e., living, asymptomatic, and green) and dry (i.e., senescent,asymptomatic, and brown) leaf material was collected from threeindividuals of each of three species of Cupressaceae (Platycladusorientalis, Cupressus arizonica and Juniperus deppeana) at theUniversity of Arizona Campus Arboretum. Leaf material was collected inlate spring to early summer from branches ca. 1.5 m above ground. Alltrees were apparently healthy and were cultivated in a park-like settingwith supplemental water.

Two mass-loss experiments were conducted. In the first preliminaryexperiment, we focused on seven endophyte/EHB associations, with inoculaprepared as above but including only the native association and axenicfungus in each case: Pestalotiopsis sp. 9143 with Luteibacter sp. 9143;Microdiploda sp. 9145 with Luteibacter sp. 9145; Cladosporium sp. 9128with Curtobacterium sp. 9128, Alternaria sp. 9055 with Sphingomonas sp.9055, Microdiplodia sp. 9145 with Erwinia sp. 9145, Microdiplodia sp.9140 with Pantoea sp. 9140, and Microdiplodia sp. 9140 with Rhizobiumsp. 9140. These associations were isolated from Platycladus orientalisin a previous study (Hoffman and Arnold 2010) and are accessioned at theRobert L. Gilbertson Mycological Herbarium at the University of Arizona(accessions MYCO-ARIZ 9128, MYCO-ARIZ 9140, MYCO-ARIZ 9143, MYCO-ARIZ9145, MYCO-ARIZ 9055). We evaluated the mass loss of green and senescenttissues of three species of Cupressaceae, listed above.

In the second experiment, we focused only on Pestalotiopsis sp. 9143 andMicrodiplodia sp. 9145. We used the eight fungal inocula describedabove, including axenic, native, resynthesized, and cross-infectedassociations, and considered green and senescent tissues of threespecies of Cupressaceae, listed above.

In each experiment, leaf samples were washed in tap water and placed in0.5 g amounts into individual, sterile 100 mm Petri plates. Three plates(experiment 1) or six plates (experiment 2) were prepared per tissuetype and sample for each inoculum type. Each leaf sample wassurface-sterilized by flooding with 95% EtOH (10 sec) followed by 0.53%NaOCI (2 min), and 70% EtOH for (2 min) (Arnold and Lutzoni 2007). Eachsample then was inoculated with 3 mL of sterile water and 75 uL offungal inoculum prepared by grinding a 6 mm plug of actively growingmycelium on 2% MEA in 1 mL sterile water. Control treatments consistedof a 6 mm plug of sterile medium ground in 1 mL of sterile water. Toevaluate whether fungi present in leaf tissue prior to inoculation couldinfluence our results, an additional trial was included in which samplesof dry material from P. orientalis were collected and autoclaved (1.2bar, 20 min) after surface-sterilization to inactivate endogenousendophytes.

Plates were wrapped three times with Parafilm, weighed immediately, andthen incubated at room temperature with approximately 12 h light/darkcycles. Plates were weighed weekly for 6 weeks. At the end of theexperiment, fungal growth was scored visually on a scale from 0 to 4(0=no visible growth, 1=1-25% coverage, 2=26-50% coverage, 3=51-75%coverage and 4=76-100% coverage of available leaf material by fungalmycelium). Mass loss was calculated as the difference in mass of eachsample after 6 weeks relative to the original mass, scaled by originalweight. Mass was predicted to leave each sample in the form of carbondioxide due to aerobic respiration and to a lesser extent, water vapor.For each fungal inoculum, mean scaled mass loss was compared by ANOVA todetermine the effects of EHB presence and identity, followed by post-hocStudent's t-tests with a Bonferroni-adjusted alpha level of 0.0083. Allanalyses were carried out in JMP v. 11.0.0 (SAS Institute, Cary, N.C.,USA).

Results

Growth on Cellulase Assay Plates

All fungal inocula, and both axenic bacteria, grew successfully oncellulase assay plates. For both Pestalotiopsis sp. 9143 andMicrodiplodia sp. 9145, colony diameter on that medium was notinfluenced significantly by the presence or absence of EHB (FIG. 2). Foreach fungal species, colony diameter on cellulase assay plates did notdiffer between resynthesized associations (i.e., the fungus and itsnative EHB reassociated after curing) and native associations with EHB.

However, as shown in FIG. 2, growth of both fungal strains wasinfluenced by the identity of EHB. Growth of Pestalotiopsis sp. 9143 wassignificantly reduced when infected by Luteibacter sp. 9145 relative toinfection by Luteibacter sp. 9143 or axenic growth. Growth ofMicrodiplodia sp. 9145 was significantly increased when infected byLuteibacter sp. 9143 relative to infection by Luteibacter sp. 9145 oraxenic growth. In both cases, fungi infected with Luteibacter sp. 9143grew more than conspecific fungal strains infected with Luteibacter sp.9145. The growth of axenic bacteria was similar between the twobacterial species.

Growth on Ligninase Assay Plates

All fungal inocula grew successfully on ligninase assay plates, as shownin Table 2 and Table 3. Neither axenic bacterium grew on this medium.

TABLE 2 Growth Activity Cellulase Ligninase Cellulase Ligninase FungusBacterium Status medium medium activity activity PestalotiopsisLuteibacter Naturally Yes Yes Yes^(A) Yes^(A) sp. 9143 sp. 9143 infectedPestalotiopsis Luteibacter Resynthesized Yes Yes Yes^(A) Yes^(A) sp.9143 sp. 9143 Pestalotiopsis Luteibacter Cross- Yes Yes Yes^(A) No sp.9143 sp. 9145 infected Pestalotiopsis None Axenic Yes Yes No Yes^(A) sp.9143 fungus Microdiplodia Luteibacter Naturally Yes Yes Yes^(a) No sp.9145 sp. 9145 infected Microdiplodia Luteibacter Resynthesized Yes YesYes^(a) No sp. 9145 sp. 9145 Microdiplodia Luteibacter Cross- Yes YesYes^(a) No sp. 9145 sp. 9143 infected Microdiplodia None Axenic Yes YesNo Yes sp. 9145 fungus None Luteibacter Axenic Yes No Yes¹ N/A sp. 9143bacterium None Luteibacter Axenic Yes No Yes¹ N/A sp. 9145 bacterium

TABLE 3 Cellulase Ligninase activity activity Fungus Bacterium StatusMean ± SD Mean ± SD Pestalotiopsis Luteibacter Naturally 1.02 ± 0.031.06 ± 0.01 sp. 9143 sp. 9143 infected Pestalotiopsis Luteibacter Re-1.01 ± 0.01 1.01 ± 0.03 sp. 9143 sp. 9143 synthesized PestalotiopsisLuteibacter Cross- 1.00 ± 0.00 0.00 ± 0.00 sp. 9143 sp. 9145 infectedPestalotiopsis None Axenic 0.00 ± 0.00 1.02 ± 0.03 sp. 9143 fungusMicrodiplodia Luteibacter Naturally 1.04 ± 0.02 0.00 ± 0.00 sp. 9145 sp.9145 infected Microdiplodia Luteibacter Re- 1.03 ± 0.01 0.00 ± 0.00 sp.9145 sp. 9145 synthesized Microdiplodia Luteibacter Cross- 1.03 ± 0.010.00 ± 0.00 sp. 9145 sp. 9143 infected Microdiplodia None Axenic 0.00 ±0.00 1.14 ± 0.06 sp. 9145 fungus None Luteibacter Axenic 1.00 ± 0.00 N/Asp. 9143 bacterium None Luteibacter Axenic 1.00 ± 0.00 N/A sp. 9145bacterium

Table 2 and Table 3 show the effects of endohyphal bacteria (EHB) onfungal growth on enzyme assay plates and the presence/absence ofcellulase and ligninase activity. Columns indicate the identity offungi, the identity of bacteria, the status of each culture, and thepresence/absence of growth and activity observed on cellulase andligninase assay plates. In Table 3, columns further indicate the meansand standard deviations of the normalized zones of clearing observed oncellulase and ligninase assay plates. In Table 2, ‘Yes’ for growthindicates that growth was observed; quantitative results are presentedin Table 3 and in FIG. 2 and FIG. 3. ‘Yes’ for activity indicatesobserved clearing of the medium; quantitative results are presented inTable 3. Within each panel, superscripts within columns are the same ifquantitative results for active strains did not differ significantly.The last two rows indicate results for axenic bacterial cultures.

For both Pestalotiopsis sp. 9143 and Microdiplodia sp. 9145, colonydiameter on indulin medium was not influenced by the presence vs.absence of EHB, nor by EHB identity. For each fungal species, colonydiameter was greater in the resynthesized association (i.e., the fungusand its native EHB re-associated after curing) and after curing andcross-inoculation with the non-native bacterium, relative to the nativeassociations or when growing axenically. Within each fungal species,growth was similar between the resynthesized and the inoculated strainsregardless of the identity or novelty of the EHB.

Presence/Absence of Cellulase Activity

The presence of EHB influenced cellulase activity. For Pestalotiopsissp. 9143, clearing of the cellulose medium was observed for all fungalinocula containing EHB, but not when the fungus was grown axenically.The same result was obtained for Microdiplodia sp. 9145. The cellulosemedium was also cleared by both bacteria when grown axenically.

Quantification of Cellulase Activity

For Pestalotiopsis sp. 9143, the identity of EHB did not significantlyinfluence the extent of clearing beyond colony edges on cellulosemedium. Clearing was consistent when the fungus was infected nativelywith Luteibacter sp. 9143, resynthesized after curing, or infected byLuteibacter sp. 9145. Similarly, for Microdiplodia sp. 9145, theidentity of EHB did not significantly influence the zone of clearingbeyond colony edges on cellulose medium.

Presence/Absence of Ligninase Activity

For Pestalotiopsis sp. 9143, clearing of the indulin medium was observedfor all fungal inocula except that containing Luteibacter sp. 9145,indicating an effect of EHB identity and an intrinsic capacity of thefungus to clear the indulin medium when growing axenically. Clearing wasonly observed for Microdiplodia sp. 9145 when it was grown axenically:the presence of EHB was associated with a loss of ligninase activity. Asmentioned above, both axenic bacteria failed to grow on the indulinmedium.

Quantification of Ligninase Activity

For Pestalotiopsis sp. 9143, the identity of EHB significantlyinfluenced the zone of clearing beyond colony edges. Clearing wassignificantly greater when the fungus was infected with its nativebacterium (Luteibacter sp. 9143, either naturally or through curing andresynthesis), or when grown axenically, than when it carried Luteibactersp. 9145 (for which no clearing was observed outside the colony edge).Clearing by the fungus with Luteibacter sp. 9143 did not differsignificantly from clearing by the fungus when grown axenically.

For Microdiplodia sp. 9145, the presence of EHB prevented clearingbeyond colony edges while the axenic strain displayed significantly moreclearing. No zone of clearing was observed when fungal inocula containedEHB, and the identity of the EHB did not influence clearing of themedium.

In Vitro Mass Loss Experiment 1

When results for seven fungal strains were considered together, thepercent of mass lost during the experiment from green foliage of P.orientalis did not differ as a function of the presence or absence ofEHB in fungal inocula (FIG. 4). Similarly, EHB did not influence massloss from green foliage of J. deppeana or C. arizonica, nor fromsenescent tissue of those species (FIG. 1). However, the presence of EHBin fungal inocula led to significantly greater mass loss from senescentfoliage of P. orientalis compared to fungi without EHB (FIG. 4).

In Vitro Mass Loss Experiment 2

In the second mass-loss experiment, no significant difference wasobserved in the percent of mass lost from fresh material of P.orientalis as a function of EHB presence or identity (FIG. 5A,Pestalotiopsis; FIG. 5B, Microdiplodia). However, mass loss differedsignificantly in senescent tissue of P. orientalis as a function of EHBtreatment within each fungal host. Significantly more mass was lost fromsenescent foliage treated with Pestalotiopsis sp. 9143 with its nativebacterium than when treated by the axenic fungus (FIG. 5C). Mass lossdue to the resynthesized association did not differ significantly fromtreatment with the native association (FIG. 5C). The presence ofLuteibacter sp. 9145 led to a decrease in mass loss relative to the samefungus containing Luteibacter sp. 9143 (FIG. 5C).

Mass loss differed significantly in senescent tissue of P. orientalistreated with Microdiplodia as a function of EHB presence, but notidentity (FIG. 5D). Mass loss was significantly greater whenMicrodiplodia contained EHB vs. treatment by the axenic fungus (FIG.5D). There was no difference in mass loss among Microdiplodia strainswith the native bacterium, following resynthesis with Luteibacter sp.9145, or following cross-infection with Luteibacter sp. 9143.

Mass loss was consistent for both fungi when they contained Luteibactersp. 9143, but not when they contained Luteibacter sp. 9145. In thatcase, mass loss was greater in the native association than in thecross-infected association (FIGS. 5C and 5D; t=5.250, df=9, P=0.0005).Results were consistent when P. orientalis tissue was autoclaved priorto the experiment (results not shown).

Each plate was scored visually for hyphal coverage at the end of theexperiment. Mass loss was positively related to the prevalence of hyphalcoverage.

Here we examined the influence of EHB on foliar fungi with a focus onsubstrate use, enzyme activity, and the ability to degrade plantmaterial. We focused specifically on cellulase and ligninase activitybecause of the central importance of such enzymes to plant cell walldecomposition and their applications in biofuel production and relatedindustries. Our study is the first to evaluate how EHB influenceenzymatic activity of fungi. Our work reveals the previously overlookedimportance of these bacterial endosymbionts in cellulase and ligninaseactivity and plant decomposition. Moreover, we show that particularbacteria have distinctive phenotypic effects, which differ as a functionof the fungus that they inhabit.

An important aspect of this work was the establishment of protocols forcuring fungal strains of their EHB, reintroducing EHB to those curedcultures, and exchanging EHB among fungal taxa. Here, we showed thatLuteibacter strains could be transferred to establish new symbioses withnovel fungal hosts in a different class of Ascomycota. Moreover, weshowed that two EHB strains that were 100% identical in their 16S rRNAhad different effects on their original and novel hosts. In general,bacterial-fungal associations that were generated by resynthesis werevery similar in phenotype to original associations (but see ligninaseresults, above).

Substrate Use

Using in vitro assays, we found that EHB influenced the ability of focalfungi to use cellulose- and lignin-based substrates. Although growth onthe cellulase assay medium did not differ as a function of the presenceand absence of EHB, the identity of the bacterial strain resulted indifferences in growth on that medium. In those assays, inoculation withLuteibacter sp. 9143 always yielded greater growth than inoculation withLuteibacter sp. 9145.

In growth assays on the ligninase assay medium, the capacity of fungi togrow was not influenced by presence, absence, or identity of EHB.Resynthesized and cross-infected strains grew more than did strains withthe native infection, and resynthesis is most effective on low-nutrientconditions. The absence of malt extract in the ligninase assay mediumresulted in it having a lower nutrient composition than the cellulaseassay medium. This may lead to enhanced fungal growth when nutrients arelimiting due to EHB presence, particularly if the bacterial titer isincreased by the resynthesis process. In future work we will test thehypothesis that the greater growth of resynthesized strains on ligninaseassay plates may reflect an increased bacterial titer relative tonaturally infected strains. We have observed the loss of EHB from fungalcultures having been repeatedly subcultured or in long-term storage(Hoffman et al. 2013), potentially contributing to a disparity in titerlevels between recultured, native associations and newly resynthesizedassociations.

EHB can Alter Fungal Enzyme Activity

EHB can alter cellulase and ligninase activity of foliar fungi in vitro.Enzyme activity may be altered by presence of EHB (cellulase) ordetermined by the genotype of the symbionts (ligninase). The increase offungal cellulase activity as a result of bacterial infection may be dueto enzyme production by the bacterium, or an increased activity due tothe symbiosis. Axenic Luteibacter sp. 9143 and 9145 grew on cellulasemedium plates, and exhibited cellulase activity under the bacterialcolonies. However, neither of the bacteria grew on ligninase mediumplates. Thus, fungal ligninase activity may be associated with a changein fungal growth as a result of EHB identity, rather than production ofligninase enzymes by the bacteria themselves.

In general, we observed that when bacterial treatment affected fungalgrowth on one medium, we saw no change or a change in growth in the samedirection on the other medium. The exception was Pestalotiopis sp. 9143inoculated with Luteibacter sp. 9145, which grew less on cellulasemedium and more on liginase medium relative to the axenic fungus or thenatural association. Pestalotiopis sp. 9143 with Luteibacter sp. 9145demonstrated cellulase activity but not ligninase activity, and thusdiffered in both assays from the axenic fungus and the naturalassociation. Because the pattern differed in Microdiplodia, our resultscan be taken to suggest that specific associations, and novelty ofinteractions, can be powerful in shaping the outcome of enzyme activity.

EHB Increased Fungal Degradation of Leaf Litter

In two experiments, EHB increased fungal degradation of, and fungalgrowth on, senescent foliage of the host species from which fungi wereoriginally isolated (P. orientalis). However, EHB did not influencedegradation of leaf litter from other confamilial trees, nor livingmaterial from any host species. In future work we will evaluate thehypothesis that specific signals produced in living foliar tissue or atthe onset of plant tissue death influence fungal/bacterial growth andactivity.

In Pestalotiopsis, we observed greater mass loss when associationsdemonstrated both ligninase and cellulase activity (vs. only ligninaseor only cellulase activity). Each Microdiplodia inoculum was only activein one enzyme, but in that species the presence or degree of cellulaseactivity was the more important indicator of potential mass loss.

Conclusions. Together, our results reveal that EHB can influenceenzymatic activity and plant biomass degradation by fungal endophytes.

Thus, described herein are methods for transferring endohyphal bacteriumfrom a first endophytic fungus to a second endophytic fungus comprisingthe following steps: preparing a bacterial inoculum of the endohyphalbacterium from the first endophytic fungus, and inoculating the secondendophytic fungus with the bacterial inoculum. In more specificembodiments, the methods comprises using first and second endophyticfungi of same species and strain. In other more specific embodiments,the first and second endophytic fungi are of different strains of thesame species. In yet other more specific embodiments, the first andsecond endophytic fungi are of different species.

Also described are methods for transferring endohyphal bacterium from afirst endophytic fungus to a second endophytic fungus comprising thefollowing steps: preparing a bacterial inoculum of the endohyphalbacterium from the first endophytic fungus, and inoculating the secondendophytic fungus with the bacterial inoculum, and wherein the first andsecond endohyphal bacteria are of the same species, or are of differentstrains of the same species, or are of different species.

Also described herein are methods for transferring endohyphal bacteriabetween a first endophytic fungus and a second endophytic funguscomprising the following steps: preparing a first bacterial inoculum ofthe endohyphal bacterium from the first fungus, preparing a secondbacterial inoculum of the endohyphal bacterium from the second fungus,and cross-inoculating the fungi by inoculating the first fungus with thesecond bacterial inoculum and inoculating the second fungus with thefirst bacterial inoculum.

Also described herein are methods for examining phenotypic changes in anendophytic fungi of the phylum Ascomyota comprising treating theendophytic fungi with an antibiotic to cure the fungi of its endohyphalbacteria, and comparing phenotypic activity of the fungi before andafter treatment with the antibiotic. In more specific embodiments, themethod comprises inoculating the cured fungi with endohyphal bacteria toresynthesize the fungi with endohyphal bacteria symbiont, and comparingthe phenotypic activity of the cured fungi to the resynthesized fungi.

Also described herein are methods for altering the enzymatic activity ofendophytic fungi, comprising transferring endohyphal bacteria from afirst endophytic fungus to a second endophytic fungus. In more specificembodiments of this method, the enzymatic activity is increased, ordecreased, or is lignocellulytic activity. In yet other embodiments, thefungus is Pestalotiopsis and the bacterium is Luteibacter, or the fungusis Microdiplodia and the bacterium is Luteibacter. In a specificembodiment, the method involves the enzymatic activity of degradingplant material.

Also described herein is a method for increasing degradation of plantmaterial comprising increasing enzymatic activity of endophytic fungi inthe plant material.

References cited in the application are listed below.

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What is claimed is:
 1. A method for examining phenotypic changes in anendophytic fungi of the phylum Ascomyota comprising treating a firstendophytic fungi with an antibiotic to produce a cured fungi by curingthe endophytic fungi of its native endohyphal bacteria, and comparingphenotypic activity of the endophytic fungi before treatment with theantibiotic to phenotypic activity of the cured fungi.
 2. The method ofclaim 1, further comprising inoculating the cured fungi with non-nativeendohyphal bacteria to resynthesize the cured fungi with endohyphalbacteria to produce a resynthesized fungi, and comparing phenotypicactivity of the cured fungi to phenotypic activity of the resynthesizedfungi.
 3. The method of claim 1, further comprising inoculating thecured fungi with non-native endohyphal bacteria to resynthesize thecured fungi with endohyphal bacteria to produce a resynthesized fungi,and comparing phenotypic activity of the first endophytic fungi tophenotypic activity of the resynthesized fungi.
 4. The method of claim1, wherein the first endophytic fungi is selected from the groupconsisting of Microdiplodia, Alternaria, Cladosporium andPestalotiopsis.
 5. The method of claim 2, wherein the first endophyticfungi is selected from the group consisting of Microdiplodia,Alternaria, Cladosporium and Pestalotiopsis.
 6. The method of claim 3,wherein the first endophytic fungi is selected from the group consistingof Microdiplodia, Alternaria, Cladosporium and Pestalotiopsis.
 7. Themethod of claim 2, wherein the non-native endohyphal bacteria isselected from the group consisting of Erwinia, Luteibacter, Sphingomonasand Burkholderia.
 8. The method of claim 3, wherein the non-nativeendohyphal bacteria is selected from the group consisting of Erwinia,Luteibacter, Sphingomonas and Burkholderia.
 9. The method of claim 1,wherein the phenotypic activity is selected from the group consisting ofcellulase activity, ligninase activity, or a combination of cellulaseand ligninase activity.
 10. The method of claim 2, wherein thephenotypic activity is selected from the group consisting of cellulaseactivity, ligninase activity, or a combination of cellulase andligninase activity.
 11. The method of claim 3 wherein the phenotypicactivity is selected from the group consisting of cellulase activity,ligninase activity, or a combination of cellulase and ligninaseactivity.